ES2222160T3 - ARABITOL PRODUCTION PROCEDURE FOR CONTINUOUS FERMENTATION. - Google Patents

Abstract

Production of arabitol (I) comprises continuous fermentation of sugar(s) in two successive fermentation zones with sugar addition to just the first zone. Production of arabitol (I) by continuous fermentation of sugar(s) with (I)-producing microorganisms comprises: (a) conducting production of (I) in a first fermentation zone (Z1), comprising at least one fermenter, in such a way that p of the sugar added to the fermentation medium is consumed by the microorganisms; (b) transferring part of the fermentation medium from Z1 to a second fermentation zone (Z2), comprising at least one fermen while maintaining constant volume in Z1 by adding sugar; (c) continuing production of (I) in Z2 so that the residual sugar in the fermentation medium is consumed; and (d) continuously separating the fermentation medium from Z2 into a concentrated microorganism fraction and a (I)-rich solub fraction.

Description

Arabitol production procedure by continuous fermentation.

The present invention relates to a Arabitol production process by continuous fermentation, at least one sugar through microorganisms producing arabitol

It has more specifically for object a Arabitol production process by continuous fermentation of at least one sugar by microorganisms producing arabitol that uses at least two fermentation zones connected to each other.

More precisely, the procedure according to the invention consists in carrying out the production of arabitol in a first fermentation zone, favoring the consumption of a part of the sugar introduced into the fermentation medium it contains, and to then transfer a part of said fermentation medium to a second fermentation zone, all of which keeping the volume of said first zone by adding sugar, and following Arabitol production in the second fermentation zone of so that residual sugar is consumed.

We proceed next, in the second zone of fermentation, to the continuous separation of the medium from fermentation with a reduced residual sugar content obtained thus in a fraction concentrated in microorganisms and another soluble fraction enriched in arabitol, and eventually at recycling of these microorganisms at the entrance of the first zone of fermentation.

The industrial preparation of arabitol is based mainly in fermentation procedures.

In general, the biological procedures of Arabitol production can use two modes of fermentation different: discontinuous and continuous.

In batch mode, they are used in a manner Classic two techniques: the technique of discontinuous fermentation proper (or batch) and fermentation technique fed batch (or fed batch).

In the technique of discontinuous fermentation proper, all the substrates necessary for the Feed of microorganisms are introduced at the beginning of the fermentation, and the arabitol produced is extracted at the end of the fermentation.

"Substrates" means the set of nutritional elements that are introduced into the fermentation medium. In the sense of the invention, the substrates are mainly those carbon sources (including sugar) and Nitrogenated directly assimilable by microorganisms arabitol producers.

This discontinuous fermentation technique it presents, however, the inconvenience of requiring time prolonged fermentation, to allow total sugar consumption introduced into the fermentation medium, and require the use of large volumes of the fermentation medium, and therefore of fermenters, to obtain conversion yields satisfactory All this translates into productivity in volume mediocre.

In addition, it happens that the variation of the concentration of one of the constituents of the fermentation medium affects performance or productivity in arabitol.

One solution is to use the second batch fermentation technique, called fermentation discontinuous fed.

This technique consists especially in introducing the substrates progressively in the fermentation medium.

The advantage of this technique is, therefore, not only to be able to control the concentration in substrates of the medium of fermentation, but also allow total sugar consumption inserted.

The fed batch fermentation technique allows, therefore, to solve the problems related to potential inhibitory effects of some of the constituents of the fermentation substrates with respect to the growth of microorganisms or the production of arabitol, or regulate, by example, more finely the aeration that conditions the Arabitol production

However, to obtain returns and satisfactory productivity, it is necessary to use high concentrations of substrates. Above all, the durations of fermentation are long and it is necessary to ensure heavy control and annoying of the different stages of fermentation.

Thus, for example, the procedure described by ESCALANTE and others, (in Journal of Fermentation and Bioengineering, 70-4, 228-231, 1990) for the production of arabitol by Hansenula in fermentation discontinuous fed, does not allow to obtain yields and industrially interesting products.

Indeed, even if arabitol is produced from Selectively, and if the batch fermentation procedure fed to ensure the consumption of all glucose introduced into the fermentation medium, arabitol is only obtained with a yield of the order of 14%.

The second fermentation mode used of Classic way for the production of arabitol by biological route is the continuous mode, as described, for example, by JAKOBUS van ZYL and PRIOR in Appl. Microbiol Biotechnol (1990), 33, 12-17

In continuous mode, all substrates of fermentation are continuously adjusted in the fermenter, and fractions are extracted from the fermentation medium with the same flow rate that the contribution of substrates, so that volume is worked constant.

This fermentation mode allows a gain appreciable performance and productivity, but does not allow obtain a high purity product, since arabitol removed is necessarily contaminated by the substrates reintroduced in the course of fermentation.

Therefore, the main drawback of Fermentation procedures in continuous mode is the need of purifying arabitol. In addition, purification techniques only allow to separate the arabitol from residual sugar in a difficult way not assimilated by microorganisms.

For this reason, it is classically associated with these continuous fermentation procedures, complex, heavy and expensive purification facilities.

All the efforts of specialists in fermentation are therefore dedicated to the investigation of operating conditions that lead to reconcile the best factors of performance and productivity in arabitol, with a reduced residual sugar content that allows a little purification expensive and easy of the arabitol produced.

The present invention therefore has the objective of solving the production problem of arabitol with a satisfactory purity and excellent yields and productivity by the use of a fermentation technique continuously specific.

The production process of arabitol used by the requesting company is to perform the fermentation of at least one sugar by microorganisms Arabitol producers in two fermentation zones.

The invention relates more particularly to a Arabitol procedure by continuous fermentation at least a sugar by arabitol producing microorganisms, which characterized by the following phases:

a) drive the production of arabitol in a first fermentation zone, which it comprises at least one fermenter, so that a part of the sugar introduced into the fermentation medium is consumed by said microorganisms,

b) transfer a part of the fermentation medium thus obtained in a second fermentation zone, which comprises at least one fermenter, keeping the volume constant in the first zone of sugar additive fermentation,

c) continuation of the production of arabitol in said second fermentation zone so that it consumes residual sugar from the medium of fermentation,

d) separation of continuous form of the fermentation medium of said second zone of fermentation thus obtained in a fraction concentrated in microorganisms and other soluble fraction enriched in arabitol,

e) collection of arabitol produced in this way.

The first stage of the procedure according to the invention consists in conducting the production of arabitol in a first fermentation zone, which comprises at least one fermenter, so that a part of the sugar introduced into the fermentation medium is consumed by said microorganisms.

The microorganisms are advantageously chosen among osmotolerant yeasts, natural or modified, producers of arabitol from sugar as a carbon source directly assimilable.

By "osmotolerant yeasts", it comprise yeasts capable of withstanding osmotic pressures important and corresponding to the genres Yamadazyma, Debaromyces, Hansenula, Candida, Zygosaccharomyces and Saccharomyces ... without this list being limiting.

By "natural osmotolerant yeasts", it comprise osmotolerant yeasts that are isolated from their environment for its natural abilities to produce a metabolite determined, in this case, arabitol.

For "modified osmotolerant yeasts", osmotolerant yeasts are understood whose natural abilities to produce a certain metabolite, in this case arabitol, have been optimized by the implementation of techniques mutagenesis (randomized or directed) or biology techniques molecular.

"Sugar" is understood herein. invention all carbon sugars directly assimilable by Arabitol producing microorganisms.

It is such a sugar, for example, that you can choose from the group consisting of glucose, galactose, sucrose, arabinose, glycerol, fructose, maltose, silulose, Ribulose, mannitol, myo-inositol, ethanol, starch and maltose, alone or in mixture with each other.

The first fermentation zone is used from way that ensures a high production of arabitol, favoring the consumption of a part of the sugar introduced in the middle of fermentation.

It is understood as "a part of sugar", of according to the invention, a minimum amount of sugar of 50% in weight, preferably at least 80% by weight, and more preferably still a minimum of 90% by weight.

In a first preferred embodiment of the method according to the invention, the production of arabitol is starts in the first fermentation zone discontinuously, simultaneously introducing all fermentation substrates, sugar and microorganisms included in them.

In a second preferred embodiment of the method according to the invention, the production of arabitol is starts in the first fermentation zone discontinuously fed, progressively feeding in sugar of the microorganisms producing arabitol.

A form of advantageously is chosen discontinuous feed type fed to start the fermentation in the process according to the invention.

This form of discontinuous feeding allows ensure the progressive filling of the first fermentation zone in substrates so that a high ratio of growth of microorganisms, a good conversion of sugar into arabitol and a sugar concentration of the fermentation medium a a value tolerated by the arabitol producing microorganism considered.

It is understood as "concentration in sugar tolerated by the microorganism, "a concentration of sugar minimum that does not inhibit the growth of said microorganism or its Arabitol production

In general, for all microorganisms producers of arabitol, the conditions used are regulated by way to avoid a total consumption of sugar.

The high production of arabitol in the first fermentation zone generally leads to the maintenance of similarly high amounts of residual sugar not consumed, whose Importance is a function of the microorganisms considered. From in general, this residual sugar ratio is at least equal to 2.5% by weight.

The second stage of the procedure according to the invention then consists in transferring a part of the means of fermentation obtained in this way in a second zone of fermentation, which comprises at least one fermenter, maintaining simultaneously the constant volume in the first zone of fermentation by adding sugar.

In this way the transfer of a part of the fermentation medium of the first fermentation zone a the second, when said fermentation medium is enriched in arabitol, but it still contains residual sugar not consumed, in quantity such that it makes the complex and heavy purification of arabitol produced.

This transfer is accompanied by a continuous feeding of sugar in the first fermentation zone, in order not only to keep the volume constant, but especially to continue the production of arabitol with a productivity and conversion performance at least equal to the values obtained in the production initiation phase of arabitol described above.

It is advantageously chosen to feed in a manner continue the first fermentation zone with the substrates, instead to do it only with sugar, as indicated in an example later.

The third stage of the procedure according to the invention consists in continuing the production of arabitol in the second fermentation zone so that sugar is consumed residual part of the fermentation medium from the First fermentation zone.

The second fermentation zone is used by Therefore simultaneously to continue the production of arabitol, but above all to end the consumption of sugar that pollutes the part of the fermentation medium transferred from the first zone of fermentation to the second, so that it only leaves a weak proportion of residual sugar.

A reduced residual sugar content is advantageously understands a concentration of sugar in the medium maximum fermentation equal to 2% by weight, and preferably maximum equal to 1% by weight.

In a preferred mode of the process according to the invention, therefore, is conducted at this stage using fermenters of different dimensions, presenting the fermenters of the second fermentation zone a lower volume to those of the first fermentation zone.

In an even more embodiment Preferred method according to the invention, no use more than one fermenter per fermentation zone.

The fourth stage of the procedure according to the invention consists in continuously separating the means of fermentation with reduced residual sugar content obtained from this way, in a fraction concentrated in microorganisms and another soluble fraction enriched in arabitol.

Continuous separation between the microorganisms and the fraction enriched in arabitol by any means known to those skilled in the art, for example, microfiltration, using membranes whose pore diameter adapts to the dimensions of the microorganism considered or by centrifugation in a range of 1,000 to 10,000 G, and so preferred by microfiltration, as indicated below with a example.

The clarified solution, enriched in arabitol, obtained at the end of this separation stage, constitutes the arabitol product.

It has also been discovered by society applicant that may be advantageous to recycle the enriched fraction in microorganisms at the entrance of the first zone of fermentation.

The advantage of this stage of recycling of microorganisms is to reintroduce a biomass whose capacities of fermentation are complete, and avoid a delay in it the implementation that would cause the preparation of a new cultivation of arabitol producing microorganisms.

The recovery of arabitol produced is effects, for example, by concentration up to a value of the order of 20% and higher, and can be continued by a stage of crystallization by any method known to the technicians in the matter.

Other features and advantages of the invention will appear from reading the non-limiting examples that are described below.

Example 1

The two fermentation zones used each one of them has a fermenter, but of dimension different. The first CHEMAP® brand fermenter has a volume of 20 l (17 l useful), and the second, of the same brand, a volume of 5 l (4 l useful).

A preculture of the Yamadazyma microorganism ohmeri, strain ATCC 20209, is made in principle in another 20 l fermenter.

The preculture medium is constituted by glucose with a concentration of 50 gr / l, corn steep liquor with a concentration of 8 gr / l, KH2PO4 at a concentration of 2 gr / l and MgSO4 with a concentration of 1 gr / l. The preculture is performed at 30 ° C, with agitation at 600 rpm, aeration of 1 vvm, for 12 hours, and a pH regulation at 4.5 with 20% NH 4 OH.

The fermenter of the first fermentation zone It is inseminated with 1 l of this preculture. It is completed at 6 l with a medium comprising glucose with a concentration of 50 gr / l, liquor of "corn steep" with a concentration of 8 gr / l, KH 2 PO 4 with a concentration of 2.5 gr / l, MgSO 4 with a concentration of 1 gr / l and FeSO4 with a concentration of 0.05 gr / l.

The temperature is set at 30 ° C. The pH is regulated at value 4.5 to 35 hours with the help of NH 4 OH 20%, and at then the regulation is set to value 3 with the help of KOH 5 N.

Glucose addition is ensured as indicated in the following table I. Biomass is estimated by measurement of the absorbance (optical density or OD) of the medium at 620 nm.

For the Yamadazyma ohmeri, a unit of absorbance at 600 nm equals a biomass of the order of 0.2 gr / l.

TABLE I

one

After 40 hours of fermentation, it is reached the useful volume of the fermenter of the first fermentation zone, and the concentration in residual glucose is 29 gr / l.

Then 4 l of the medium is transferred fermentation of this first fermenter in the fermenter of the second fermentation zone, and then 800 g of glucose are added in a volume of 2 l in the first fermenter. The temperature of the second fermentation zone is set at 38 ° C, and the pH is regulated at 3.5 with the help of KOH 5 N.

The fermenters evolve next separately up to 46 hours, after which the first extraction of arabitol in the second fermenter.

After 46 hours, the fermenter of the first fermentation zone is fed in glucose with a concentration of 250 gr / l with a flow rate of the order of 0.4 l / h and in "corn steep" liquor with a flow rate of 50 gr / l with a flow rate of the order of 10 ml / h.

The half circle of the first zone of second fermentation using a PCM® pump, and then from the second fermentation zone to a filter module Tangential MEMBRALOX®, which allows to recover a permeate rich in arabitol and a retained rich in microorganisms.

The detainee is then reintroduced to the entrance of the first fermentation zone.

To keep the levels in both fermenters, the volume of the liquid resulting from the filtrate Outgoing is equal to the feed rate in glucose.

This flow evolves over time in function of the speed of glucose consumption by microorganisms in the fermenters. After 200 hours, the feeding stops, and the second zone fermenter Fermentation is emptied.

The fermentation is allowed to finish in the fermenter of the first fermentation zone for 10 hours supplementary, before tracing the medium in the filtration module tangential.

Table II attached below presents the measurements made after the transfer of the fermentation medium of the first fermentation zone to the second.

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TABLE II

2

Overall, 8.8 kg of arabitol have been recovered for 18.7 kg of glucose used, that is, a yield of 47% and a productivity of 2.2, gr / l / h.

In discontinuous fermentation carried out in same conditions of means of production, with the same contribution of glucose, the yield is 40% and the productivity is only 1.5 gr / l / h.

The process according to the invention allows for therefore ensure higher productivity returns.

In addition, the reduced glucose ratio residual allows to recover an arabitol that has a very purity satisfactory

Example 2

The preparation of arabitol is carried out in fermentation conditions identical to those of example 1, but using the microorganism Yamadazyma ohmen, strain ATCC 20209 modified by the technique of classical random mutagenesis by ultraviolet rays (using at least one cycle of mutagenesis with ultraviolet rays with niastine enrichment), the strain having been selected according to its ability to produce arabitol with the yield of 8 to 10 points higher than the strain matrix.

After obtaining a preculture of said modified strain, and transfer to the first fermentation zone as indicated in example 1, the glucose addition It is insured as presented in the following table III.

TABLE III

3

After 40 hours of fermentation, it is reached the useful volume of the fermenter of the first fermentation zone, and the concentration in residual glucose is 28 gr / l.

Then 4 l of medium of fermentation of this first fermenter to the second fermenter fermentation zone, and 800 g of glucose are then added in a 2 l volume in the first fermenter.

The temperature of the second fermentation zone it is set at 38 ° C, and the pH is regulated at 3.5 with the help of 5N KOH.

The fermenters evolve next separately up to 46 hours, time from which time performs the first arabitol extraction in the second fermenter

From 46 hours, the fermenter of the first fermentation zone is fed in glucose to a concentration of 250 gr / l with a flow rate of the order of 0.4 l / h, and in "corn steep" liquor at 50 gr / l with a flow rate of the order of 10 ml / h

Table IV attached below presents the measurements made after transfer of the fermentation medium of the first fermentation zone to the second.

TABLE IV

4

Globally, 12 kg of arabitol are recovered for 19 kg of glucose used, that is, a yield of 63% and a productivity of 4 gr / l / h.

In discontinuous fermentation carried out in same conditions of means of production, with the same contribution of glucose, the yield is 55% and the productivity is only 2.5 gr / l / h.

The process according to the invention allows, by Therefore, ensure better yields and better productivity.

In addition, the reduced glucose ratio residual always allows to recover an arabitol that has a Purity completely satisfactory.

Example 3

The two fermentation zones are used as in Example 1

Initially a preculture of Candida polymorpha microorganism, strain ATCC 20213 natural, in a CHEMAP® 20 l fermenter, under the same conditions as for the strain Y. ohmeri of example 1.

The fermenter of the first fermentation zone It is inseminated with 1 l of this preculture.

It is completed up to 6 l with a medium containing glucose with a concentration of 50 gr / l, of yeast extract a a concentration of 3 gr / l, KH 2 PO 4 at a concentration of 2 gr / l and MgSO4 at a concentration of 1 gr / l.

The temperature is set at 30 ° C. The pH is regulated to 4.5 to 30 hours with the help of NH 4 OH 20%, and then with the help of KOH 5 N.

The addition of glucose is controlled as it is indicated in the following table III.

Biomass is calculated by measuring the absorbance (optical density) of the medium at 620 nm.

For Candida polymorpha, a unit of absorbance at 600 nm equals a biomass of the order of 0.22 gr / l.

TABLE V

5

At 40 hours of fermentation, the useful volume of the first zone fermenter, and the concentration of residual glucose is 30 gr / l.

4 l of fermentation medium of this first fermenter are then transferred to the fermenter of the second fermentation zone, and 800 grams of glucose are added in a 2 l volume in the first fermenter.

The temperature of the second fermentation zone it is set at 37 ° C, and the pH is adjusted to 4.5 by 5 N KOH.

The fermenters evolve next separately up to 46 h, after which the first one is made Arabitol extraction in the second fermenter.

From 46 hours, the fermenter of the First fermentation zone is fed in glucose at 250 gr / l with a flow of the order of 0.4 l / h, and the "corn steep" liquor at 50 gr / l with a flow rate of the order of 10 ml / h.

The following table IV presents the measurements carried out after transfer of the fermentation medium of the First fermentation zone to the second.

TABLE VI

6

Overall, 4.1 kg of arabitol was recovered for 17.4 kg of glucose used, that is, a yield of 23.6% and a productivity of 1 gr / l / h.

In discontinuous fermentation carried out in same conditions of means of production, with the same contribution of glucose, the yield is 16.5% and the productivity is only 0.6 gr / l / h.

The method according to the invention allows This way, for a considered strain, ensure better yields and productivity than the classically used processes.

Moreover, the reduced glucose production residual allows to recover an arabitol that presents in this way a very satisfactory purity.

Claims (8)

1. Procedure for the production of arabitol by continuous fermentation of at least one sugar by arabitol-producing microorganisms, characterized by the fact that the following phases are carried out:

a) driving Arabitol production in a first fermentation zone, which it comprises at least one fermenter, so that a part of the sugar introduced into the fermentation medium is consumed by said microorganisms,

b) transfer of a part of the fermentation medium thus obtained in a second fermentation zone, comprising at least one fermenter, while maintaining the constant volume in the first fermentation zone by sugar additive,

c) continuation of the production of arabitol in said second fermentation zone so that residual sugar is consumed from the medium of fermentation,

d) separation of continuous form of the fermentation medium of said second zone of fermentation obtained in this way in a concentrated fraction of microorganisms and other soluble fraction enriched in arabitol,

e) collection of arabitol obtained.

2. Method according to claim 1, characterized in that said fraction concentrated in microorganisms is recycled at the entrance of the first fermentation zone. 3. Method according to one or the other of claims 1 and 2, characterized in that the part of sugar consumed by the arabitol producing microorganisms in the first fermentation zone is at least 50% by weight, preferably a minimum of 80% in weight, and more preferably still a minimum of 90% by weight of the sugar introduced into the fermentation medium. 4. Method according to one or other of claims 1 and 3, characterized in that the residual sugar content in the second fermentation zone is a maximum of 2% by weight, preferably a maximum of 1% by weight. 5. Method according to any of claims 1 to 4, characterized in that the fermenters of the second fermentation zone have a lower volume than those of the fermenters of the first zone. Method according to claim 5, characterized in that the first and second fermentation zones each comprise a single fermenter. Method according to any one of claims 1 to 6, characterized in that the arabitol producing microorganisms are chosen from the group consisting of natural or modified osmotolerant yeasts of the genus Yamadazyma, Debaromyces, Hansenula, Candida, Zygosaccharomyces and Saccharomyces. Method according to any one of claims 1 to 7, characterized in that the sugar is chosen from the carbon sources directly assimilable by arabitol-producing microorganisms, and is preferably glucose.